Cytokine deprivation from activated T cells leads to apoptosis associated with down-regulation of the bcl-2 gene product. It is not clear, however, how cytokines other than interleukin-2 (IL-2) may affect this process and regulate the involvement of other apoptosis-modulating genes. We show that a group of cytokines including IL-2 (IL-2R gamma), prevent the apoptosis of IL-2-deprived activated T cells. This rescue involves the induction of the anti-apoptosis genes bcl-2 and bcl-xL), but causes little change in expression of bax and bcl-xS, which promote apoptosis. Furthermore, the prevention of apoptosis and induction of proliferation by the common gamma chain cytokines can be dissociated. Thus, when proliferation is blocked, the common gamma chain cytokines still induce up-regulation of bcl-2 relative to bax and retard apoptosis. These cytokines can thus regulate the persistence or removal of effector T cells by coordinating the balance between genes which promote and those which inhibit apoptosis, events which are probably mediated at least in part by signals through the common gamma chain. These data also implicate inappropriate T cell apoptosis resulting from a dysfunctional common gamma-chain as part of the pathophysiological defect in patients with X-linked severe-combined immunodeficiency (SCID).

The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly upregulated following p53 induction. Pifithrin ␣, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21 CIP1 . Surprisingly, pifithrin ␣ dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein.

Summary It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric α/α form are more cytotoxic than their CD8– counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8– NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8α+ cells after lysis of the same targets. We report that cross‐linking of the CD8α chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK‐cell apoptosis from which CD8α+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK‐cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti‐MHC Class I antibody. Thus, in contrast to the CD8– subset, CD8α+ NK cells are capable of sequential lysis of multiple target cells.

We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD 50 for PTL was 6.2 lM (n ¼ 78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1-3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34 þ haematopoietic progenitor cells. The mechanism of cell killing was via PTLinduced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IjB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.

IntroductionOnce thought to be a benign disease, chronic lymphocytic leukemia (CLL) is now known to comprise an aggressive subset, characterized by unmutated immunoglobulin heavy-chain variable (IGHV) genes, high expression of cell-surface CD38, and of the protein tyrosine kinase, zeta-chain-associated protein kinase 70 . 1 The proliferative compartment in CLL is located in "pseudofollicles" within bone marrow and lymphoid tissue, 2 and access of CLL cells to these compartments is dependent on migratory chemokine signals, including chemokine (C-X-C motif) ligand 12 (CXCL12), 2 chemokine (CC motif) ligand 19 (CCL19), and CCL21. 3 CLL is treated with chlorambucil or fludarabine, but is incurable by chemotherapy alone. 4 Because drug resistance is a formidable obstacle to treatment, it is important to identify and evaluate the mechanisms that contribute to apoptosis resistance in CLL and to develop strategies to overcome them.Conventional DNA-damaging cytotoxic agents, including fludarabine and chlorambucil, kill CLL cells via the induction of the proapoptotic p53 protein. 5 Therefore, mutations or deletions affecting the p53 genes are a major cause of drug resistance. 6 However, genetic changes involving p53 are selected for during treatment, and this type of resistance is acquired over time. 6 In contrast, environmental factors mediate drug resistance from the initiation of treatment, resulting in the protection of tumor cells from diverse chemotherapeutic agents and in the survival of foci of residual disease. 2,7 It is therefore desirable to develop strategies that can be applied during initial treatment and are designed to minimize the survival of malignant cells and the consequent emergence of acquired resistance.CLL cells, especially those located in bone marrow or lymphoid tissue, are exposed to extracellular signals that promote their survival and proliferation through the suppression of apoptotic pathways. 2 Interleukin-4 (IL-4) is a Th2 lymphokine that signals via receptor-mediated activation of the Janus protein tyrosine kinases, JAK1 and JAK3, and subsequent phosphorylation and activation of the signal transducer and activator of transcription 6 (STAT6) transcription factor. 8 IL-4 prolongs the basal survival of CLL cells in vitro, 9 and the IL-4 receptor is abundantly expressed by CLL cells, compared with normal B lymphocytes, correlating with the substantially greater antiapoptotic effect of IL-4 on CLL cells. 10,11 Extensive evidence suggests that IL-4/CLL interactions occur in vivo as well. First, an IL-4-secreting CD3 ϩ /CD8 ϩ /CD28 Ϫ T-lymphoid population is expanded in CLL. 12 Second, CLL cells secrete IL-6, which switches T lymphocytes from a T-helper 1 (Th1)-to an IL-4-secreting Th2 phenotype. 13 Third, 40%-65% of CD8 ϩ T cells of CLL patients contained IL-4, while Ͻ 10% of these cells from normal subjects did so. 14 These T cells were also able to secrete IL-4 without activation. Fourth, the proportion of IL-4-positive T cells was greater in patients with progressive disease, compared with st...

Activation of the phosphatidylinositol 3-kinase/AKT pathway antagonizes apoptosis in diverse cellular systems. We previously showed that human plasma activated AKT and potently blocked the ability of chlorambucil or gamma radiation to induce apoptosis of B-chronic lymphocytic leukemia (

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The A-helical amphipathic peptide D-(KLAKLAK) 2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC 50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/ peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.